2012 Annual Report 1a.Objectives (from AD-416): 1. Develop a “reverse search” tool, which would allow a sequence database generated by a massively parallel sequencing (MPS) run to be searched with a limited number of key pathogen sequences. This will cut processing time exponentially, increasing efficiency and throughput. 2. Test MPS diagnostic capacity on select plant pathogens from viruses, phytoplasmas, bacteria, fungi and stramenopiles. 3. Test MPS forensic (strain typing) capacity on select plant pathogens, where previous typing data is available. 4. Test the ability of MPS to detect plant pathogens that have been genetically engineered to express toxins or proteins harmful to humans, by screening plant pathogen DNA samples spiked with examples of such genes or commonly used genetic engineering promoters and/or plasmid screening markers. 1b.Approach (from AD-416): Massively parallel sequencing (MPS), commonly referred to as “454 sequencing” or pyrosequencing, generates tremendous numbers of overlapping short sequence reads that are then compiled by computer to generate huge contigs of sequence. The most common use of MPS is to generate genomic sequences, but the technology can also be used to create a diagnostic assay with the capacity to detect and potentially strain type any and all pathogens in a sample. In this collaborative project, between USDA-ARS FDWSRU and the Oklahoma State University National Institute for Microbial Forensics and Food and Agricultural Biosecurity, we will test the the capacity of MPS to act as a forensic tool, and potentially allow the screening of DNA samples for the hallmarks of genetically engineered harmful toxins/proteins. In the preliminary phase, we will apply 454 FLX MPS to generate a sequence sample database (SSD) from 16 test pathogens (representing viruses, phytoplasmas, bacteria, fungi and stramenopiles), and identify a minimum of 5 highly diagnostic sequences for each of the 16 target pathogens. Target sequences will be identified (roughly 100 nucleotides in length) that are conserved among strains and isolates of the pathogen, and not found in the genomes of the other test pathogens genomes of plants or of frequently occurring plant endosymbionts for which sequence information is available. We will then test forensic (strain typing) capacity on select plant pathogens, where previous typing data is available. In the second phase, we will test the ability of MPS to detect plant pathogens that have been genetically engineered to express toxins or proteins harmful to humans, by screening plant pathogen DNA samples spiked with examples of such genes or commonly used genetic engineering promoters and/or plasmid screening markers. The collaboration between USDA-ARS and Oklahoma State University National Institute for Microbial Forensics and Food and Agricultural Biosecurity will also present unique opportunities for education and outreach. OSU will use their grant funds to send NIMFAB students to Fort Detrick to train on the latest techniques and equipment with scientists at FDWSRU and the National Bioforensics Analysis Center on the Fort Detrick National Interagency Biodefense Campus. A workshop bringing together experts from academia, government and industry labs will be planned for the final year of the grant. 3.Progress Report: The objective of this project was to determine if massively parallel sequencing (MPS) was a viable technique for the detection of plant pathogens. Initial work using simulated MPS databases determined that the approach used was successful at detecting pathogens, even when pathogen sequences made up as little as 0.5 percent of the total sequence reads. An optimal query length was determined, and modeling demonstrated that MPS based techniques could successfully detect RNA viruses, DNA viruses, spiroplasmas, bacteria, oomycetes and fungi. Subsequent work with infected plant sequencing has demonstrated that the approach is successful in planta for fungi and viruses, but not bacteria.